Genetically encoded ratiometric biosensor for probing lysosomal pH in mammalian cells and C. elegans

ABSTRACTLysosomes are important sites for macromolecular degradation, defined by a profoundly acidic lumenal pH of ~4.5. To better understand lysosomal pH, we designed a novel, genetically encoded, fluorescent protein-based pH biosensor called FIRE-pHLy (Fluorescence Indicator REporting pH in Lysosomes). This sensor was targeted to lysosomes with LAMP1 and reported lumenal pH between 3.0 and 6.0 with monomeric teal fluorescent protein 1 (mTFP1), a bright cyan FP variant with a pKa of 4.3. Ratiometric quantification was enabled with cytosolically oriented mCherry using high-content imaging. We expressed FIRE-pHLy in several cellular models as well as the multicellular organism C. elegans and quantified its alkalinizing response to bafilomycin A1, a specific V-ATPase inhibitor. In summary, we have engineered FIRE-pHLy, a specific, robust and versatile lysosomal pH biosensor that has broad applications for investigating pH dynamics in aging and lysosome-related diseases, as well as in lysosome-based drug discovery.

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Acidocalcisomes in Toxoplasma gondii tachyzoites

Biochemical Journal ◽

10.1042/bj3130655 ◽

1996 ◽

Vol 313 (2) ◽

pp. 655-659 ◽

Cited By ~ 106

Author(s):

Silvia N. J. MORENO ◽

Li ZHONG

Keyword(s):

Endoplasmic Reticulum ◽

Toxoplasma Gondii ◽

Mammalian Cells ◽

Specific Inhibitor ◽

Mitochondrial Atpase ◽

Bafilomycin A1 ◽

Atpase Inhibitor ◽

Extracellular Medium ◽

Acidic Compartment ◽

Acidic Organelles

Toxoplasma gondii tachyzoites were loaded with the fluorescent indicator fura 2 to investigate the transport mechanisms involved in maintaining their intracellular Ca2+ homoeostasis. The mitochondrial ATPase inhibitor oligomycin and the endoplasmic-reticulum Ca2+-ATPase inhibitor thapsigargin increased the intracellular Ca2+ concentration ([Ca2+]i), thus indicating the requirement for ATP and the involvement of the endoplasmic reticulum in maintaining intracellular Ca2+ homoeostasis. The effect of thapsigargin was more accentuated in the presence of extracellular Ca2+, clearly showing that, as occurs with other eukaryotic cells, depletion of intracellular Ca2+ pools led to an increase in the uptake of Ca2+ from the extracellular medium. In addition to these results, we found evidence that, in contrast with what occurs in mammalian cells, T. gondii tachyzoites possess a significant amount of Ca2+ stored in an acidic compartment, termed the acidocalcisome, as indicated by: (1) the increase in [Ca2+]i induced by bafilomycin A1 (a specific inhibitor of H+-ATPases), nigericin (a K+/H+ exchanger) or the weak base NH4Cl, in the nominal absence of extracellular Ca2+ to preclude Ca2+ entry; and (2) the effect of ionomycin, a Ca2+-releasing ionophore that cannot take Ca2+ out of acidic organelles and that was more effective after alkalinization of these compartments by addition of bafilomycin A1, nigericin or NH4Cl. Considering the relative importance of the ionomycin-releasable and the ionomycin+NH4Cl-releasable Ca2+ pools, it is apparent that T. gondii tachyzoites contain a significant amount of Ca2+ stored in acidocalcisomes.

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Quantitative assessment of near-infrared fluorescent proteins

10.21203/rs.3.rs-797586/v1 ◽

2021 ◽

Author(s):

Kiryl Piatkevich ◽

Hanbin Zhang ◽

Stavrini Papadaki ◽

Xiaoting Sun ◽

Luxia Yao ◽

...

Keyword(s):

Mammalian Cells ◽

Quantitative Assessment ◽

Near Infrared ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Oligomeric State ◽

C Elegans ◽

The Right ◽

Infrared Fluorescent Protein

Abstract Recent progress in fluorescent protein development has generated a large diversity of near-infrared fluorescent proteins, which are rapidly becoming popular probes for a variety of imaging applications. To assist end-users with a selection of the right near-infrared fluorescent protein for a given application, we will conduct a quantitative assessment of intracellular brightness, photostability, and oligomeric state of 19 near-infrared fluorescent proteins in cultured mammalian cells. The top-performing proteins will be further validated for in vivo imaging of neurons in C. elegans, zebrafish, and mice. We will also assess the applicability of the selected NIR FPs for expansion microscopy and two-photon imaging.

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Quantitative characterization of extracellular vesicle uptake and content delivery within mammalian cells

Nature Communications ◽

10.1038/s41467-021-22126-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Emeline Bonsergent ◽

Eleonora Grisard ◽

Julian Buchrieser ◽

Olivier Schwartz ◽

Clotilde Théry ◽

...

Keyword(s):

Mammalian Cells ◽

Fluorescent Protein ◽

Extracellular Vesicle ◽

Content Delivery ◽

Bafilomycin A1 ◽

Quantitative Characterization ◽

Endosomal Acidification ◽

A Cell ◽

Dose Responses

AbstractExtracellular vesicles (EVs), including exosomes, are thought to mediate intercellular communication through the transfer of cargoes from donor to acceptor cells. Occurrence of EV-content delivery within acceptor cells has not been unambiguously demonstrated, let alone quantified, and remains debated. Here, we developed a cell-based assay in which EVs containing luciferase- or fluorescent-protein tagged cytosolic cargoes are loaded on unlabeled acceptor cells. Results from dose-responses, kinetics, and temperature-block experiments suggest that EV uptake is a low yield process (~1% spontaneous rate at 1 h). Further characterization of this limited EV uptake, through fractionation of membranes and cytosol, revealed cytosolic release (~30% of the uptaken EVs) in acceptor cells. This release is inhibited by bafilomycin A1 and overexpression of IFITM proteins, which prevent virus entry and fusion. Our results show that EV content release requires endosomal acidification and suggest the involvement of membrane fusion.

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Function of a STIM1 Homologue in C. elegans: Evidence that Store-operated Ca2+ Entry Is Not Essential for Oscillatory Ca2+ Signaling and ER Ca2+ Homeostasis

The Journal of General Physiology ◽

10.1085/jgp.200609611 ◽

2006 ◽

Vol 128 (4) ◽

pp. 443-459 ◽

Cited By ~ 39

Author(s):

Xiaohui Yan ◽

Juan Xing ◽

Catherine Lorin-Nebel ◽

Ana Y. Estevez ◽

Keith Nehrke ◽

...

Keyword(s):

Mammalian Cells ◽

Fluorescent Protein ◽

Contractile Activity ◽

C Elegans ◽

Unfolded Protein ◽

Acid Protein ◽

Ef Hand ◽

Green Fluorescent ◽

Oscillations And Waves ◽

Soc Channels

1,4,5-trisphosphate (IP3)-dependent Ca2+ signaling regulates gonad function, fertility, and rhythmic posterior body wall muscle contraction (pBoc) required for defecation in Caenorhabditis elegans. Store-operated Ca2+ entry (SOCE) is activated during endoplasmic reticulum (ER) Ca2+ store depletion and is believed to be an essential and ubiquitous component of Ca2+ signaling pathways. SOCE is thought to function to refill Ca2+ stores and modulate Ca2+ signals. Recently, stromal interaction molecule 1 (STIM1) was identified as a putative ER Ca2+ sensor that regulates SOCE. We cloned a full-length C. elegans stim-1 cDNA that encodes a 530–amino acid protein with ∼21% sequence identity to human STIM1. Green fluorescent protein (GFP)–tagged STIM-1 is expressed in the intestine, gonad sheath cells, and spermatheca. Knockdown of stim-1 expression by RNA interference (RNAi) causes sterility due to loss of sheath cell and spermatheca contractile activity required for ovulation. Transgenic worms expressing a STIM-1 EF-hand mutant that constitutively activates SOCE in Drosophila and mammalian cells are sterile and exhibit severe pBoc arrhythmia. stim-1 RNAi dramatically reduces STIM-1∷GFP expression, suppresses the EF-hand mutation–induced pBoc arrhythmia, and inhibits intestinal store-operated Ca2+ (SOC) channels. However, stim-1 RNAi surprisingly has no effect on pBoc rhythm, which is controlled by intestinal oscillatory Ca2+ signaling, in wild type and IP3 signaling mutant worms, and has no effect on intestinal Ca2+ oscillations and waves. Depletion of intestinal Ca2+ stores by RNAi knockdown of the ER Ca2+ pump triggers the ER unfolded protein response (UPR). In contrast, stim-1 RNAi fails to induce the UPR. Our studies provide the first detailed characterization of STIM-1 function in an intact animal and suggest that SOCE is not essential for certain oscillatory Ca2+ signaling processes and for maintenance of store Ca2+ levels in C. elegans. These findings raise interesting and important questions regarding the function of SOCE and SOC channels under normal and pathophysiological conditions.

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The lysosomotrope GPN mobilises Ca2+from acidic organelles

Journal of Cell Science ◽

10.1242/jcs.256578 ◽

2021 ◽

Vol 134 (6) ◽

pp. jcs256578 ◽

Cited By ~ 1

Author(s):

Yu Yuan ◽

Bethan S. Kilpatrick ◽

Susanne Gerndt ◽

Franz Bracher ◽

Christian Grimm ◽

...

Keyword(s):

Acute Treatment ◽

Chronic Treatment ◽

The Novel ◽

Bafilomycin A1 ◽

Atpase Inhibitor ◽

Cytosolic Ph ◽

Lysosomal Ph ◽

Lysosomotropic Agent ◽

Acidic Organelles ◽

Primary Action

ABSTRACTLysosomes are acidic Ca2+stores often mobilised in conjunction with endoplasmic reticulum (ER) Ca2+stores. Glycyl-L-phenylalanine 2-naphthylamide (GPN) is a widely used lysosomotropic agent that evokes cytosolic Ca2+signals in many cells. However, whether these signals are the result of a primary action on lysosomes is unclear in light of recent evidence showing that GPN mediates direct ER Ca2+release through changes in cytosolic pH. Here, we show that GPN evoked rapid increases in cytosolic pH but slower Ca2+signals. NH4Cl evoked comparable changes in pH but failed to affect Ca2+. The V-type ATPase inhibitor, bafilomycin A1, increased lysosomal pH over a period of hours. Acute treatment modestly affected lysosomal pH and potentiated Ca2+signals evoked by GPN. In contrast, chronic treatment led to more profound changes in luminal pH and selectively inhibited GPN action. GPN blocked Ca2+responses evoked by the novel nicotinic acid adenine dinucleotide phosphate-like agonist, TPC2-A1-N. Therefore, GPN-evoked Ca2+signals were better correlated with associated pH changes in the lysosome compared to the cytosol, and were coupled to lysosomal Ca2+release. We conclude that Ca2+signals evoked by GPN most likely derive from acidic organelles.

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Antifungal Activity Directed Toward the Cell Wall by 2-Cyclohexylidenhydrazo- 4-Phenyl-Thiazole Against Candida albicans

Infectious Disorders - Drug Targets ◽

10.2174/1871526518666180531101605 ◽

2019 ◽

Vol 19 (4) ◽

pp. 428-438 ◽

Cited By ~ 1

Author(s):

Nívea P. de Sá ◽

Ana P. Pôssa ◽

Pilar Perez ◽

Jaqueline M.S. Ferreira ◽

Nayara C. Fonseca ◽

...

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Antifungal Activity ◽

Mammalian Cells ◽

C Elegans ◽

Buccal Epithelial Cells ◽

Mutant Strains ◽

Low Toxicity ◽

High Concentrations ◽

Mic Value

Background: The increasing incidence of invasive forms of candidiasis and resistance to antifungal therapy leads us to seek new and more effective antifungal compounds. Objective: To investigate the antifungal activity and toxicity as well as to evaluate the potential targets of 2- cyclohexylidenhydrazo-4-phenyl-thiazole (CPT) in Candida albicans. Methods: The antifungal activity of CPT against the survival of C. albicans was investigated in Caenorhabditis elegans. Additionally, we determined the effect of CPT on the inhibition of C. albicans adhesion capacity to buccal epithelial cells (BECs), the toxicity of CPT in mammalian cells, and the potential targets of CPT in C. albicans. Results: CPT exhibited a minimum inhibitory concentration (MIC) value of 0.4-1.9 µg/mL. Furthermore, CPT at high concentrations (>60 x MIC) showed no or low toxicity in HepG2 cells and <1% haemolysis in human erythrocytes. In addition, CPT decreased the adhesion capacity of yeasts to the BECs and prolonged the survival of C. elegans infected with C. albicans. Analysis of CPT-treated cells showed that their cell wall was thinner than that of untreated cells, especially the glucan layer. We found that there was a significantly lower quantity of 1,3-β-D-glucan present in CPT-treated cells than that in untreated cells. Assays performed on several mutant strains showed that the MIC value of CPT was high for its antifungal activity on yeasts with defective 1,3-β-glucan synthase. Conclusion: In conclusion, CPT appears to target the cell wall of C. albicans, exhibits low toxicity in mammalian cells, and prolongs the survival of C. elegans infected with C. albicans.

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Split-wrmScarlet and split-sfGFP: Tools for faster, easier fluorescent l abeling of endogenous proteins in Caenorhabditis elegans

Genetics ◽

10.1093/genetics/iyab014 ◽

2021 ◽

Author(s):

Jérôme Goudeau ◽

Catherine S Sharp ◽

Jonathan Paw ◽

Laura Savy ◽

Manuel D Leonetti ◽

...

Keyword(s):

Fluorescent Protein ◽

Fluorescent Proteins ◽

Fluorescent Labeling ◽

Large Fragment ◽

C Elegans ◽

High Affinity ◽

Red Fluorescent Proteins ◽

Invaluable Tool ◽

Endogenous Proteins ◽

Fluorescent Tags

Abstract We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous C. elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663

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Enhanced brightness of bacterial luciferase by bioluminescence resonance energy transfer

Scientific Reports ◽

10.1038/s41598-021-94551-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Tomomi Kaku ◽

Kazunori Sugiura ◽

Tetsuyuki Entani ◽

Kenji Osabe ◽

Takeharu Nagai

Keyword(s):

Energy Transfer ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Color Change ◽

Yellow Fluorescent Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Bioluminescence Resonance Energy Transfer ◽

Bacterial Luciferase ◽

Bacterial Bioluminescence

AbstractUsing the lux operon (luxCDABE) of bacterial bioluminescence system as an autonomous luminous reporter has been demonstrated in bacteria, plant and mammalian cells. However, applications of bacterial bioluminescence-based imaging have been limited because of its low brightness. Here, we engineered the bacterial luciferase (heterodimer of luxA and luxB) by fusion with Venus, a bright variant of yellow fluorescent protein, to induce bioluminescence resonance energy transfer (BRET). By using decanal as an externally added substrate, color change and ten-times enhancement of brightness was achieved in Escherichia coli when circularly permuted Venus was fused to the C-terminus of luxB. Expression of the Venus-fused luciferase in human embryonic kidney cell lines (HEK293T) or in Nicotiana benthamiana leaves together with the substrate biosynthesis-related genes (luxC, luxD and luxE) enhanced the autonomous bioluminescence. We believe the improved luciferase will forge the way towards the potential development of autobioluminescent reporter system allowing spatiotemporal imaging in live cells.

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Functional expression and characterization of the C. elegans G-protein-coupled FLP-2 Receptor (T19F4.1) in mammalian cells and yeast

International Journal for Parasitology Drugs and Drug Resistance ◽

10.1016/j.ijpddr.2012.10.002 ◽

2013 ◽

Vol 3 ◽

pp. 1-7 ◽

Cited By ~ 8

Author(s):

Martha J. Larsen ◽

Elizabeth Ruiz Lancheros ◽

Tracey Williams ◽

David E. Lowery ◽

Timothy G. Geary ◽

...

Keyword(s):

G Protein ◽

Mammalian Cells ◽

Functional Expression ◽

C Elegans ◽

G Protein Coupled

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ADP-Ribosylation Factor (ARF) Interaction Is Not Sufficient for Yeast GGA Protein Function or Localization

Molecular Biology of the Cell ◽

10.1091/mbc.e02-02-0078 ◽

2002 ◽

Vol 13 (9) ◽

pp. 3078-3095 ◽

Cited By ~ 30

Author(s):

Annette L. Boman ◽

Paul D. Salo ◽

Melissa J. Hauglund ◽

Nicole L. Strand ◽

Shelly J. Rensink ◽

...

Keyword(s):

Membrane Trafficking ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Protein Localization ◽

Carboxypeptidase Y ◽

Minor Role ◽

Temperature Sensitive ◽

Adp Ribosylation ◽

And Function ◽

Adp Ribosylation Factor

Golgi-localized γ-ear homology domain, ADP-ribosylation factor (ARF)-binding proteins (GGAs) facilitate distinct steps of post-Golgi traffic. Human and yeast GGA proteins are only ∼25% identical, but all GGA proteins have four similar domains based on function and sequence homology. GGA proteins are most conserved in the region that interacts with ARF proteins. To analyze the role of ARF in GGA protein localization and function, we performed mutational analyses of both human and yeast GGAs. To our surprise, yeast and human GGAs differ in their requirement for ARF interaction. We describe a point mutation in both yeast and mammalian GGA proteins that eliminates binding to ARFs. In mammalian cells, this mutation disrupts the localization of human GGA proteins. Yeast Gga function was studied using an assay for carboxypeptidase Y missorting and synthetic temperature-sensitive lethality between GGAs andVPS27. Based on these assays, we conclude that non-Arf-binding yeast Gga mutants can function normally in membrane trafficking. Using green fluorescent protein-tagged Gga1p, we show that Arf interaction is not required for Gga localization to the Golgi. Truncation analysis of Gga1p and Gga2p suggests that the N-terminal VHS domain and C-terminal hinge and ear domains play significant roles in yeast Gga protein localization and function. Together, our data suggest that yeast Gga proteins function to assemble a protein complex at the late Golgi to initiate proper sorting and transport of specific cargo. Whereas mammalian GGAs must interact with ARF to localize to and function at the Golgi, interaction between yeast Ggas and Arf plays a minor role in Gga localization and function.

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